WO1998026798A2

WO1998026798A2 - IMMUNIZATION AGAINST NEISSERIA GONORRHOEAE AND $i(NEISSERIA MENINGITIDIS)

Info

Publication number: WO1998026798A2
Application number: PCT/US1997/023755
Authority: WO
Inventors: Robert J. Arko; Cheng-Yen Chen; Stephen A. Morse; David Trees
Original assignee: Arko Robert J; Chen Cheng Yen; Morse Stephen A; David Trees
Priority date: 1996-12-19
Filing date: 1997-12-19
Publication date: 1998-06-25
Also published as: AU5617698A; WO1998026798A3

Abstract

The present invention relates to a method of immunization against infection by a pathogenic Neisseria species. The method employs parenteral priming using a priming angiten, followed by oral immunization with gamma-irradiated, Protein III-deficient killed whole cells of Neisseria gonorrhoeae strain 340 or parenteral priming using a priming antigen followed by oral immunization with gamma-irradiated class 4 protein-deficient whole cells of Neisseria meningitidis. Also provided is a method for protecting a human patient against infection by N. meningitidis and N. gonorrhoeae comprising orally administering to the patient a protective amount of a class 4 protein-deficient, killed whole cell of N. meningitidis. In addition, the present invention provides a method for protecting a human patient against infection by N. meningitidis and N. gonorrhoeae comprising orally administering to the patient a protective amount of a class 4 protein-deficient, killed whole cell of N. meningitidis and an oral adjuvant.

Description

IMMUNIZATION AGAINST NEISSERIA GONORRHOEAE AND NEISSERIA MENINGITIDIS

BACKGROUND OF THE INVENTION

This invention was made with government support funded by the Centers for Disease Control and Prevention through a grant from the National Vaccine Program. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for inducing immunity to infection by Neisseria gonorrhoeae and/or Neisseria meningitidis. In particular, the present invention relates to an oral whole cell immunization method.

BACKGROUND ART

Neisseria gonorrhoeae is the causative agent of gonorrhea, an extremely common infection in humans, prevalent in the United States as well as in other countries. Heretofore, no successful vaccine against the numerous strains of N. gonorrhoeae has been developed. While several vaccine preparations and immunization methods have been proposed [U.S. Patent Nos. 4,443,431 (Buchanan et al), 4,220,638 (Karkhanis et al), 4,203,971 (Buchanan) and 4,681,761 (Mietzner et al)], none have provided effective protection in appropriate experimental models or in human trials.

These patents claim either (1) an antigenic complex of the cell surface

(Karkhanis et al, Buchanan); (2) an immunogenic fragment of pili protein (Buchanan et al); or (3) a purified major iron regulatory protein (MIRP) (Mietzner et al). Methods in all of these patents describe parenteral administration of the vaccine component. Although all of these patents address the issues of cross-reactivity, none deals effectively with development of ucosal immunity, the blocking effects of antibodies against Protein III (PHI), or toxicity problems inherent in parenteral vaccines.

These factors are likely to be significant, considering that previous human vaccine trials employing surface antigens on cells and pilus proteins have failed to demonstrate a successful immunoprotective response to gonococcal challenge, especially with heterologous strains (Boslego, J.W. & Deal, CD., (1991), in Vaccines and Immunotherapy, (S.J. Cruz, ed.), pp.21 1-224, Pergamon Press, New York, Boslego etal, (1991), Vaccine, 9: 154-162).

The state of the technology in gonococcal vaccine development has been refined to focus on individual cell surface proteins such as Protein I (PI) and MIRP. However, despite numerous trials utilizing a parenteral PI vaccine, a protective response has not been generated (Boslego & Deal (1991), Gulati et al, in Neisseriae, 1991, pp.229-234, Walter de Guyter & Co., Berlin). In addition, although MIRP has shown potential as a vaccine agent, data disclosed in Table 3 herein and in Tables 1, 2 and 4 in the parent application demonstrate that the parenteral administration of this protein alone fails to induce a protective response to gonococcal challenge.

N. gonorrhoeae has efficiently evolved as a sexually transmitted disease which eludes the human body's immune mechanisms quite effectively and requires new immunization strategies. Thus, there remains a great need for a vaccine against N. gonorrhoeae in order to prevent the spread of this infectious agent, for which treatment is becoming more difficult due to the development of multiple resistance to antibiotics.

A related organism, Neisseria meningitidis, is a causative agent of septicemia and bacterial meningitis. The latter is a central nervous system infection most commonly afflicting small children with significant morbidity and mortality. N. meningitidis is a very common pharyngeal organism that can penetrate mucosal tissues and cause systemic disease especially in individuals suffering from various immune deficiencies including defects in late acting complement components. Sporadic epidemics also occur under conditions of close physical contact that promote aerosol transmission. A large geographical area of sub-Saharan Africa is regularly affected by outbreaks of meningitis and has become known as the "meningitis belt" (Martin & Cremieux, in The Pathogenic Neisseria, 1984, pp.608-610). Approximately 3000 cases of meningococcal disease occur annually in the U.S. with around 150 deaths (Morbidity and Mortality Weekly Report, Summary of Notifiable Diseases, United States, 43:3, 1974). The rapidity with which the disease can strike apparently healthy individuals including young adults creates great fear in communities where clustering of cases has occurred and often produces increased pressure on local officials for initiation of intervention programs even though the current vaccines available for this purpose are expensive to administer and of questionable value. Although numerous strategies, similar to those used in development of vaccines against N. gonorrhoeae, have been employed in attempts to produce an effective N. meningitidis vaccine, there remains a need for an immunization protocol which affords broader and more long lasting protection, especially in early childhood, to reduce the economic burden and morbidity caused by these organisms.

Although capsule-specific polysaccharide vaccines are available for some of the meningococcal serogroups, limitations due to the T-cell independent nature of these vaccines and the absence of a group B vaccine reduce their effectiveness, especially in younger age groups. Routine meningococcal immunization is not generally practiced in the U.S. and sporadic disease claims a number of lives each year. The medical and economic impact of these diseases even in the more developed countries is considerable; however, the devastation and suffering caused in emerging areas of the world is of a much greater magnitude.

The challenge of controlling the pathogenic neisseria remains formidable due to an increasing variety of antimicrobial resistance factors that can be genetically transferred among a number of mucosal organisms (Morbidity and Mortality Weekly Report, CDC Sentinel Surveillance for Antimicrobial Resistance in N. gonorrhoeae, 43:29-39, 1993). This is made even more critical by the absence of any highly effective vaccine for inducing long-term immunity. Thus, a critical need exists for an oral meningococcal vaccine that also protects against gonorrhea and which is therefore likely to be better accepted by society than would a strictly gonococcal vaccine, thus providing better utilization of limited health resources.

This invention satisfies these needs by providing a method which is effective in preventing infection by N. gonorrhoeae and N. meningitidis. The present invention provides a method of immunization against pathogenic Neisseria species which overcomes problems previously encountered by using conventional parenteral immunization methods, i.e., induction of "blocking" antibodies, failure of the host to respond to conformation epitopes found on intact micro-organisms, development of a long-lasting mucosal immune response and problems associated with toxicity due to contamination with endotoxin.

SUMMARY OF THE INVENTION

The present invention provides an effective method of inducing immunity in humans against N. gonorrhoeae that is protective against a number of different gonococcal strains and an effective method of inducing immunity in humans against N. meningitidis. Also provided is an effective method of inducing immunity in humans that is protective against a number of different strains of both N. gonorrheae and N. meningitidis.

The present invention provides a method of immunization against N. gonorrhoeae. The method comprises parenteral administration of a priming antigen (e.g. a synthetic immunoadjuvant, for example, polyphosphazene, or a synthetic peptide from N gonorrhoeae Protein IB with the amino acid sequence DDQTYSIPSLFV (SEQ ID NO: 1), QHQNYSIPSLFN (SEQ ID ΝO:2), EHQVYSBPSLFV (SEQ ID NO.3) or ASVAGTNTGWGNK (SEQ ID NO:4), or a combination of these peptides) in a pharmaceutically acceptable carrier to a human subject, in an amount sufficient to enhance the immune response to subsequently administered oral doses of a gonococcal immunogen. The oral immunogen can consista of Pill-deficient, killed whole cells of N. gonorrhoeae in a pharmaceutically acceptable carrier, in an amount sufficient to induce resistance to infection with N. gonorrhoeae.

In addition, the present invention provides a method of immunization against N. meningitidis. The method comprises parenteral administration of a priming antigen in a pharmaceutically acceptable carrier to a human subject in an amount sufficient to enhance an immune response to subsequently administered oral doses of an immunogen. The oral immunogen can consist of killed protein class 4-deficient whole cells ofN. meningitidis in a pharmaceutically acceptable carrier, in an amount sufficient to induce resistance to infection with N. meningitidis.

Furthermore, the present invention provides various kits. The kits comprise a first container with a priming antigen or an oral adjuvant in a pharmaceutically acceptable carrier suitable for parenteral administration and a second container with the oral whole cell component in a pharmaceutically acceptable carrier suitable for oral administration.

Also provided in the present invention is a method for protecting a human patient against infection by N. meningitidis and N. gonorrhoeae comprising orally administering to the patient a protective amount of a class 4 protein-deficient_^ killed whole cell of N. meningitidis.

Furthermore, the present invention provides a method for protecting a human patient against infection by N. meningitidis and N. gonorrhoeae comprising orally administering to the patient a protective amount of a class 4 protein-deficient_^ killed whole cell of N. meningitidis and an oral adjuvant. Additionally provided in the present invention is a kit comprising a container with aclass 4 protein-deficient, killed whole cell of N. meningitidis in a pharmaceutically acceptable carrier for oral administration.

Also provided in the present invention is a method for protecting a human patient against infection by Neisseria meningitidis and Neisseria gonorrhoeae comprising parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response and subsequently orally administering a protective amount of a class 4 protein- deficient, killed whole cell of Neisseria meningitidis.

A method for protecting a human patient against infection by Neisseria meningitidis and Neisseria gonorrhoeae is also provided, comprising parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response and subsequently orally administering a protective amount of a Protein Ill-deficient killed whole cell of Neisseria gonorrhoeae.

Various other objectives and advantages of the present invention will become apparent from the following description.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples included herein. As used in the claims, "a" can include multiples.

The present invention provides a method of protecting a patient against infection by N. gonorrhoeae comprising the steps of parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response and subsequently orally administering a protective amount of a Protein Ill-deficient killed whole cell ofN. gonorrhoeae or a whole cell of wild type N. gonorrhoeae. Also provided in the present invention is a method of protecting a human patient against infection by N. gonorrhoeae comprising parenterally administering to the patient a priming synthetic immunoadjuvant in an amount sufficient to induce an immune response and subsequently orally administering a protective amount of a Protein Ill-deficient killed whole cell ofN. gonorrhoeae. The synthetic immunoadjuvant can be polyphosphazene and the Protein Ill-deficient killed whole cell ofN. gonorrhoeae can be produced from strain 340, deposited with the American Type Culture Collection under the accession number ATCC 55320.

According to the method of the present invention, a human subject is first primed with a priming antigen, which can be, for example, a synthetic immunoadjuvant (e.g. polyphosphazene), one or more immunogenic peptides of gonococcal PI, or other priming antigens determined to be effective in the present invention. The effectiveness of the priming antigens can be determined according to the methods described in Examples 4 and 5.

By "synthetic immunoadjuvant" is meant an agent which enhances an immune response and consists of an organic polymer which can contain inorganic moieties which confer additional properties, such as solubility and flexibility. Examples of synthetic immunoadjuvants which do not contain inorganic moieties are poly (lactide-co- glycolide) (Stolle-DuPont Company, Cincinnati, Ohio) and Carbopol (B. F. Goodrich, Cincinnati, Ohio). An example of a synthetic immunoadjuvant with inorganic moieties is polyphosphazene (Virus Research Institute, Cambridge, Massachusetts), an organic polymer with phosphorous atoms in its backbone structure. An example of a polyphosphazene in the present invention is a water soluble, ionically-crosslinked molecule that forms with molecular weight of 3-4 million and is thus not excreted when injected parenterally but is slowly hydrolyzed to ammonium phosphate and phosphoric acid to effect its removal from tissue. In addition, the synthetic immunoadjuvant for the present invention can be tucaresol (589C80) [chemical name: 4-(2-formyl-3- hydroxyphenoxymethyl) benzoic acid] (Glaxo Wellcome). Examples of immunogenic peptides include, but are not limited to, those having or consisting essentially of the following amino acid sequences: DDQTYSIPSLFV (SEQ ID NO. l), EHQVYSIPSLFV (SEQ ID NO:3), QHQVYSDPSLFV (SEQ ID NO:2) and ASVAGTNTGWGNK (SEQ ID NO:4). By "consisting essentially of is meant a peptide having the amino acid sequence disclosed including minor substitutions, additions or deletions which do not negatively effect the immunogenicity of the peptide.

Immunogenic peptides ofN. gonorrhoeae can be obtained or synthesized using standard methods known in the art, for example, by computer based predictive algorithms and automated peptide synthesis (Rothbard & Taylor, 1989, EMBO 7:93- 100). The purity of the peptides can be determined by analytical HPLC and the presence of endotoxin can be determined with a rabbit pyrogen test. The effectiveness of an immunogenic peptide as a priming antigen in the present invention can be determined according to the methods set forth in the Examples herein.

The priming antigen is parenterally administered in an amount sufficient to induce an immune response which imparts a priming effect (Hosmalin et al, J. Immunol, 146: 1667- 1673 (1991)). In the present invention, the synthetic peptide is parenterally administered in a pharmaceutically acceptable carrier to human subjects. Suitable carriers for use in the present invention include, but are not limited to, pyrogen- free water. For parenteral administration of the priming antigen, a sterile solution or suspension is prepared in water that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected, for example, into subcutaneous or intramuscular tissues.

Between 100-400 μg or approximately 1-5 μg/kg of the priming antigen can be administered in three injections at two week intervals or a single human dose of 800 μg (10 μg/kg) can be given. However, the type of priming antigen, as well as the age, weight and condition of the individual must also be considered in determining a final dose. Alternatively, the priming antigen may be microencapsulated using natural or synthetic polymers. Although one skilled in the art will realize that dosages are best determined by the practicing physician, dependent on the individual patient, one intramuscular injection of 800 μg of the microencapsulated priming antigen can be sufficient to generate the desired response.

The second step of the immunization method of the present invention involves orally administering a protective amount of a gonococcal antigen, for example, killed (e.g. gamma-irradiated) Pill-deficient (PUT) whole cells ofN. gonorrhoeae (for example, strain 340). N. gonorrhoeae strain 340 has been shown to induce a high level of cross-protection to different gonococcal strains when used as a formaldehyde-killed parenteral whole cell immunogen in a mouse or guinea pig infection model. PUT mutants are preferred because their use results in a greater level of protection. A PET mutant of strain 340 was obtained by insertional inactivation of the rmp structural gene by methods known to the art, although it should be understood that other PHI^" mutants can be generated by one skilled in the art using the method taught by Wetzler et al. (J. Exp. Med, 169:2199-2209 (1989)). Briefly, a cloned rmp gene, inactivated by insertion of a ermC (erythromycin resistance) gene, was integrated into the gonococcal chromosome, generating the PHI^' phenotype.

Specifically, the PHI^" mutant was produced as follows. The cloning and sequencing of the PHI gene has been described (J. Exp. Med. 164:868 (1986); J. Exp. Med. 165:471 (1987)). The PHI λgtl 1 clone 33 was treated with Eco RI to excise the PHI insert. The insert was ligated into Eco Rl-cut pMOB45 (Gene (Amst.) 15:319 (1981)) and tetracycline-resistant, chloramphenicol-sensitive transformants were selected and isolated. A particular isolate was designated pMOB45/33-2 and was used for further constructions. The βla gene is contained on a 2.2-kb Bam HI fragment of plasmid pFA3 (J. Bacteriol. 154:1498 (1983)). The plasmid was purified fromE. coli and this fragment was isolated by restriction enzyme digestion, agarose gel electrophoresis and electroelution of the restriction enzyme fragment. Approximately 2.5 μg of pMOB45/33-2 was digested with Xba I. A unique PHI Xba I site is located at base pair 601, two-thirds into the ORF of the PHI gene. Approximately 1 μg of the purified βla Bam HI fragment was added to Xba I-treated pMOB45/33-2 and the 5' over-hanging ends left by the restriction enzyme digestions were filled using the Klenow fragment of DNA polymerase to obtain blunt ends. The fragments were ligated with T4 DNA ligase and transformed into competent cells of E. coli strain DH5.

Four colonies were selected that grew on medium containing tetracycline and carbenicillin and plasmids were isolated from these isolates, respectively designated A- D. Restriction mapping with Eco RI digestion indicated plasmids A, C and D gave rise to the expected fragments. Further restriction mapping using Pst I and Pvu I gave rise to identical patterns for the plasmids A and C but different ones from plasmid D, indicating that plasmid D contained the βla gene in the opposite orientation than that of plasmids A and C. It was concluded that the βla insert is in the same direction as the PHI gene in plasmid D but is in the reverse direction in plasmid A and C. Plasmids C and D were isolated in large quantities and then further purified by CsCl equilibrium density gradient centrifugation. Aliquots were methylated using Hae III methylase according to the vendor's protocol (New England Biolabs Inc., Beverly, MA). This step was performed to protect the DNA from one gonococcal restriction enzyme, Ngo π, which is an isoschizomer of Hae III (J. Bacteriol 155: 1324 (1983)). The plasmids were then treated with Eco RI to release the modified gonococcal DNA fragment and used for transformation.

The transformations were carried out as follows. Competent piliated gonococci of strain F62 were grown for 16-18 h on gonococcal agar (Infect. Immun. 19:320 (1978)) in a 6% CO₂ incubator at 37°C and resuspended in 1.5% Proteose Peptone No. 3 (Difco Laboratories, Detroit, MI) broth with 30mM Hepes, pH 7.2, 10mM MgCl₂, 1% Isovitalex (GC-Hepes). The resuspended organisms were diluted to a concentration of 5 X 10⁷ colony forming units ( CFU) per ml (OD^ = 0.05). Two μg of either plasmid C or D, Hae III methylated and cut with Eco RI, were added to one ml of the diluted organisms and incubated statically at 37°C. After one hour, three ml of GC-Hepes were added and the tubes with the organism were rotated at 30 rpm for 5-6 h at 37°C. Control tubes were treated identically, but no DNA was added. Various dilutions of the organisms incubated with the DNA (+DNA) and organisms without DNA (control) were spread on GC agar, overlapping a concentration gradient of ampicillin. The 11 gradient was established by concentrically spreading 50 μl of a 1 mg ml dilution of ampicillin to the middle two-thirds of each agar plate. After 48 h, the plates were examined and healthy, transparent, piliated colonies that appeared at the edge of the ampicillin gradient on plates that were inoculated with +DNA organisms were selected and propagated on GC agar containing 3 μg/ml of ampicillin. Such colonies did not appear on control plates. The isolates that grew on the ampicillin plates were then tested with nitrocefin disks (BBL Microbiology Systems, Cockeysville, MD) to detect βla activity and colonies that tested positive were further investigated. Transformations were also performed using gonococcal strains Pgh 3-2 and UU1, in a similar manner as above, using total chromosomal DNA or electrophoretically isolated restriction fragments of chromosomal DNA using transformants obtained from the above experiment. Chromosomal DNA was obtained using the method of Nakamura et al. (J. Bacteriol. 137:595 (1979)).

Oral administration of a whole cell immunogen permits vaccination with essential antigens which may be too toxic to be given parenterally. In addition, the oral route can be more effective in stimulating the mucosal immunity required to prevent gonococcal colonization. Between 1x10^s and 8xl0⁹ CFU of the oral immunogen can be administered. Although one skilled in the art will appreciate that specific immunization schedules are best designed for the needs of individual patients, an effective oral regimen of the present invention can include as many as four to ten oral doses of gonococcal antigen administered at approximately one week intervals. Oral immunization can be initiated as early as two weeks after parenteral priming or may be delayed for up to four weeks. This two to four week period between parenteral priming and oral immunization appears to be an optimal time period.

Suitable carriers for gonococcal antigens used for oral administration include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants. Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans. Suitable liquid carriers may be water, pharmaceutically accepted oils, 12 or a mixture of both. The liquid can also contain other suitable pharmaceutical additions such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents. Examples of suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a pH-regulated gel. The gonococcal antigen may be contained in enteric coated capsules that release antigens into the intestine to avoid gastric breakdown.

Alternatively, the gonococcal antigen may be microencapsulated with either a natural or a synthetic polymer into microparticles 4-8 μm in diameter, which target intestinal or vaginal lymphoid tissues and produce a sustained release of antigen for up to four weeks (Eldridge et al, Cur. Topics inMicrobiol and Immunol, 146:59-65 (1989); Oka e/α/., Vaccine, 8:573-576 (1990)).

In addition, the present invention provides a kit comprising a first container with a priming antigen in a pharmaceutically acceptable carrier for parenteral administration and a second container with a protective amount of a Protein Ill-deficient killed whole cell ofN. gonorrhoeae in a pharmaceutically acceptable carrier for oral administration. The priming antigen of the kit can be, for example, polyphosphazene or an immunogenic peptide ofN. gonorrhoeae (e.g., a peptide from N. gonorrhoeae Protein I, including the peptides of SEQ ID ΝOS: 1, 2, 3 and/or 4). The kit of the present invention can also contain more than one peptide. Also, the whole cell of the kit can be a Protein πi- deficient killed whole cell ofN gonorrhoeae produced from N. gonorrhoeae strain 340, deposited with the ATCC under the accession number ATCC 55320.

In another embodiment, the present invention provides a method of protecting a patient against infection by N. meningitidis comprising the steps of parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response against the protein and subsequently orally administering a protective amount of a killed class 4 protein-deficient whole cell ofN. meningitidis. Alternatively, the wild type killed whole cell ofN. meningitidis can be administered as the oral immunogen. According to the method of the present invention, a human subject can first be primed with either a synthetic immunoadjuvant (e.g. polyphosphazene), one or more synthetic peptides of meningococcal class 2,3 protein, or any other suitable priming antigen. The priming antigen is parenterally administered in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response which imparts a priming effect. The immunogenicity of the priming antigens can be determined according to the methods described in Examples 4 and 5.

Other priming antigens suitable for use in this invention can include, but are not limited to, Haemophilus inβuenzae b conjugate vaccine (Hib-dt, diphtheria toxoid conjugate, ProHIBit®, Connaught Laboratories, Inc., Swiftwater, PA), the pneumococcal vaccine Pneumo-23 (Merck and Co., Inc.), and the NspA protein (Plant et al. abstract, Tenth Annual International Neisseria Conference, September 8-13, 1996, Baltimore, MD).

Immunogenic peptides ofN. meningitidis can be obtained or synthesized using standard methods known in the art, for example, by computer based predictive algorithms and automated peptide synthesis (Rothbard & Taylor, 1989, EMBO 7:93- 100). The purity of the peptides can be determined by analytical HPLC and the presence of endotoxin can be determined with a rabbit pyrogen test. The effectiveness of an immunogenic peptide as a priming antigen in the present invention can be determined according to the methods set forth in the Examples herein.

The second step of the immunization method of the present invention involves orally administering a protective amount of a meningococcal antigen [for example, class 4 protein-deficient, killed (e.g. gamma-irradiated) whole cells ofN. meningitidis].

Preparation of synthetic peptides and microencapsulation protocols as described for the N. gonorrhoeae vaccine are similarly applicable to the N. meningitidis vaccine. The described examples of pharmaceutically acceptable carriers for both parenteral and oral administration for the N. gonorrhoeae vaccine can be extended to the N. meningitidis vaccine as well.

In addition, the present invention provides a kit comprising a first container with a priming antigen in a pharmaceutically acceptable carrier for parenteral administration and a second container with a protective amount of a class 4 protein deficient killed whole cell ofN. meningitidis in a pharmaceutically acceptable carrier for oral administration. The priming antigen of the kit can be, for example, polyphosphazene or an immunogenic peptide ofN. meningitidis (e.g., a peptide from theN. meningitidis Class 2 protein or Class 3 protein). The kit of the present invention can also contain more than one peptide.

A kit comprising a first container with a priming synthetic immunoadjuvant in a pharmaceutically acceptable carrier suitable for parenteral administration and a second container with a protective amount of a Protein Ill-deficient killed whole cell ofN. gonorrhoeae in a pharmaceutically acceptable carrier suitable for oral administration is also provided. The priming synthetic immunoadjuvant of the kit can be polyphosphazene and the Protein Ill-deficient killed whole cell ofN. gonorrhoeae of the kit can be produced from strain 340, deposited with the American Type Culture Collection under the accession number ATCC 55320.

The present invention also provides a method for protecting a human patient against infection by either N. meningitidis and N. gonorrhoeae, comprising orally administering to the patient a protective amount of a class 4 protein-deficient, killed whole cell ofN. meningitidis in a pharmaceutically acceptable carrier. The class 4 protein-deficient killed whole cell ofN. meningitidis can be produced from, for example, N. meningitidis strain FAM-18 (CDC). Alternatively, the wild type killed whole cell of N. meningitidis can be administered as the oral immunogen. In addition, a combination ofN. meningitidis strain FAM-18 cells and N. gonorrhoeae strain 340 cells, either wild type or Class 4 protein deficient or PIII protein deficient, respectively, can be used in the present invention. The present invention also provides a method for protecting a human patient against infection by Neisseria meningitidis and Neisseria gonorrhoeae comprising parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response and subsequently orally administering either a protective amount of a class 4 protein- deficient, killed whole cell of Neisseria meningitidis and/or a protective amount of a Protein Ill-deficient killed whole cell of Neisseria gonorrhoeae.

The N. meningitidis whole cell of the present invention can be made class 4 protein-deficient by methods known in the art, e.g., the method described by Klugman et al. (Infection and Immunity 57:2066-2071 (1989)). Alternatively, the class 4 protein- deficient cells of N. meningitidis can be produced by any other molecular genetic protocol for deletion or inactivation of the Class 4 protein gene, now known or later developed in the art.

Specifically, as described in Klugman et al., plasmid pBR322 was isolated from E. coli and phenol extracted. Plasmid DΝA was cut with Pvu II and Cla I; the ends were blunted with Klenow fragment of DΝA polymerase (Boehringer Mannheim) and ligated overnight at room temperature with T4 DΝA ligase (New England Biolabs, Inc., Beverly, MA). PvuII-Clal deletion mutants of the plasmid were selected from E. coli transformant strains that retained resistance to 50 μg of carbenicillin per ml but were now susceptible to tetracycline (a PvuII-Clal deletion of pBR322 deleted the tetracycline resistance gene on the plasmid). The plasmid was then cut with EcoRI, treated with calf intestinal alkaline phosphatase (Boehringer Mannheim) and ligated to the DNA insert (L3) containing the open reading frame of the class 4 protein structural gene (designated rmpM) derived from a λgtl 1 clone. Transformants containing either single copies or tandem repeats of rmpM were identified. The L3 insert has only a single PvuII site (nine nucleotides from the start of the DNA encoding the leader peptide of the pro-class 4 protein molecule) and a single Clal site in the DNA immediately following the termination codon of rmpM, making it possible to selectively delete rmpM from this plasmid construction. (The previous PvuII-Clal deletion of pBR322 eliminated these sites from the plasmid itself.) Following the PvuII-Cla I deletion of rmpM, the ends of the plasmid were blunted by using Klenow DNA polymerase, and a blunt-ended erythromycin resistance gene (ermO) not previously used as a marker in neisserial genetics, was ligated into the plasmid construction. The ermC gene of Bacillus subtilis is a constitutively expressed gene that differs from ermC (originally described in Staphylococcus aureus) in the coding frame by only five amino acid residues and that acts by specifying an S-adenosylmethionine-dependent 23 S rRNA transmethylase. Transformants in E. coli DH5 cells (Bethesda Research Laboratories, Inc., Gaithersburg, MD) were identified by resistance to both 50 μg of carbenicillin and 100 μg of erythromycin per ml. Finally, four μg of plasmid DNA from these transformants was treated with Hae III methlyase in the presence of S- adenosylmethionine to methylate the DNA prior to Εco RI cutting and transformation.

The N. meningitidis whole cells of the present invention can be killed, for example, by gamma irradiation. In particular, gamma irradiation can be applied to N. meningitidis whole cells at a dosage between 220 and 2500 Grays (Gy). Although complete inactivation of meningococcal cells at a concentration of 10⁹ CFU/ml was accomplished at 220 Gy, the optimal dose of irradiation for preparing the whole cells was determined to be 550 Gy (see Table 12).

The present invention further provides a method for protecting a human patient against infection by either N. meningitidis and N. gonorrhoeae comprising orally administering to the patient a protective amount of a class 4 protein-deficient, killed whole cell ofN. meningitidis, in a pharmaceutically acceptable carrier and an oral adjuvant in a pharmaceutically acceptable carrier. By "oral adjuvant" is meant an agent which enhances an immune response and can be taken orally without toxic or other undesirable side effects. The oral adjuvant can be, but is not limited to, lithium chloride (Sigma Chemical Co.), at about 5 mg per dose of whole cell immunogen; E. coli labile toxin (LT) (Bema Products Corporation, Coral Gables, FL), at about 10 μg per dose of whole cell immunogen; taurine (2-amino ethanesulfonic acid) (Sigma Chemical

Company, St. Louis, MO), at about 5 mg per dose of whole cell immunogen; tucaresol (589C80) [chemical name: 4-(2-formyl-3-hydroxyphenoxymethyl) benzoic acid] (Glaxo Wellcome), at about 10-20 mg/kg per dose of whole cell immunogen; parotin (Teikoku Hormone Mfg. Co., Tokyo; Ishizaka et al, 1990. Vaccine 8:337-341); lithium carbonate (Sigma Chemical Co., ST. Louis, MO); the NspA protein, as well as any other oral adjuvants now known or discovered in the future to enhance the immunoprotective effect of class 4 protein-deficient killed whole cells ofN. meningitidis against infection by N. meningitidis and N. gonorrhoeae.

The oral whole cell immunogen of the present invention can also be a C4 deficient N. meningitidis killed whole cell conjugated to the C3d complement peptide (Sigma Chemical Co., St. Louis, MO).

The oral whole cell immunogen of the present invention can also by a C4 deficient Ν. meningitidis killed whole cell which has been genetically engineered to overexpress the ΝspA protein. These cells can be produced according to genetic engineering protocols well known in the art (see, e.g, Sambrook et al, Molecular Cloning: A Laboratory Manual, latest edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

Oral administration of an N. meningitidis whole cell immunogen permits vaccination with essential antigens which may be too toxic to be given parenterally. In addition, the oral route can be more effective in stimulating the mucosal immunity required to prevent meningococcal and gonococcal colonization. Between lxl 0⁹ and 8xl0⁹ CFU of the N. meningitidis oral immunogen can be administered.

Although one skilled in the art will appreciate that specific immunization schedules are best designed for the needs of individual patients, an effective oral regimen of the present invention can include as many as four to ten oral doses ofN meningitidis whole cells administered at approximately one week intervals. The oral adjuvant can be administered as a single dose at the time of initial administration of the whole cell immunogen, or it can be administered prior to or following administration of the whole cell immunogen, either as a single initial dose or at intervals matching the administration of the whole cell immunogen.

Immunity enhancing amounts of the oral adjuvant can be determined using standard procedures, as described in the Examples herein. Briefly, various doses of the oral adjuvant are administered to a subject in addition to the whole cell immunogen and the immunity enhancing response of each dose is determined according to the protocols set forth in the Examples herein (see also, e.g., Arnon, R. (Ed.) Synthetic Vaccines 1:83- 92, CRC Press, Inc., Boca Raton, Florida, 1987). The exact dosage of the oral adjuvant and killed whole cells may vary from subject to subject, depending on various parameters, such as, for example, the type of oral adjuvant administered, the age, weight and condition of the subject, etc. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine screening given the teachings herein.

Generally, the dosage of the oral adjuvant will approximate that which is typical for the administration of vaccines, as determined according to protocols well known in the art. The effects of the administration of the oral adjuvant can be determined following the initial administration which can precede or be simultaneous with the administration of the whole cell oral immunogen and monitored thereafter at regular intervals as needed, for an indefinite period of time.

Suitable pharmaceutical carriers for oral administration ofN. meningitidis whole cells and oral adjuvants include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants. Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans. Suitable liquid carriers may be water, pharmaceutically accepted oils, or a mixture of both. The liquid can also contain other suitable pharmaceutical additions such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents. Examples of suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a pH-regulated gel. The N. meningitidis whole cell and/or the oral adjuvant may be contained in enteric coated capsules that release antigens into the intestine to avoid gastric breakdown.

Alternatively, the N. meningitidis whole cell and/or the oral adjuvant may be microencapsulated with either a natural or a synthetic polymer into microparticles 4-8 μm in diameter, which target intestinal or vaginal lymphoid tissues and produce a sustained release for up to four weeks (Eldridge et al, Cur. Topics in Microbio and Immunol, 146:59-65 (1989); Oka e/ cr/., Vaccine, 8:573-576 (1990)).

Also provided in the present invention is a kit comprising a first container with a class 4 protein-deficient, killed whole cell ofN. meningitidis in a pharmaceutically acceptable carrier for oral administration. A kit comprising a first container with a class 4 protein-deficient, killed whole cell ofN. meningitidis in a pharmaceutically acceptable carrier for oral administration and a second container with an oral adjuvant in a pharmaceutically acceptable carrier for oral administration, is also provided. The oral adjuvant can be lithium chloride.

The subcutaneous chamber model in mice is a well accepted model in the art and is used extensively to test functional immunity (Genco and Arko, 1994. In Methods in Enzymology 235: 120-140, Abelson and Simon, Academic Press). Although neisserial infections are studied in this model in tissue devoid of any mucosal membrane, the site has full exposure to humoral and cellular components of the host's immune system. Results obtained by the subcutaneous method have been compared with results of parallel studies of groups of orally immunized mice challenged intravaginally with N. gonorrhoeae. These results showed a good correlation between the two methods for accessing the response to oral immunization. There are a number of additional reasons for using the subcutaneous chamber method for testing of neisserial vaccines, including the ability to challenge in a noncontaminated site having porous access to the host immune response. In addition, the challenge dose 50% (DD₅₀), level of chamber colonization and clearance rate for challenge organisms can be determined under laboratory conditions that allow frequent access to clinical specimens. Because infections can be established in nonimmunized controls with relative few organisms (<200 CFU), large comparative differences can be determined for well immunized animals. Infections that might occur in immunized chambers are usually of a much shorter duration than for controls, allowing these chambers to be rechallenged with other test strains to determine levels of cross-protection. Because the connective tissues surrounding the subcutaneous chamber express receptors for gamma/delta T-cells, the chamber can amplify the effects attributable to this effector cell type (White et al., 1994. J. Immunol 152:4912-4918). In addition, the subcutaneous chamber method is conducted without the need for immunosuppressive drugs such as the estradiol used in the vaginal clearance model. When properly performed with well designed chambers, the technique can be used in mice causing them little pain or discomfort and because it does not require death as an endpoint it is more acceptable to institutional animal care and use committee review.

The method of the present invention overcomes several problems common in the art. In particular, the present invention provides an immunization method which provides protection against all of the meningococcal strains, including Group B, in addition to protection against N. gonorrhoeae. In addition, the administration of the mutant cells described in the present invention can reduce the induction of blocking antibodies to PIII from N. gonorrhoeae or to class 4 protein from N. meningitidis, which inhibit the bactericidal antibodies directed against other cell surface antigens. Furthermore, unlike conventional parenteral immunization methods, the present invention provides for an immunologic response to conformational epitopes on intact organisms. This is achieved by using intact cells for oral immunization. In comparison with parenteral subunit vaccines (e.g., pili, MIRP or PI), the whole cell oral vaccine presents a variety of antigens inducing more cross-reactive antibodies (Table 3) that are less likely to be rendered ineffective by a single mutation of the organism's surface structure. The conformational epitopes of cell envelope antigens, necessary for generating antibodies and other non-specific immune mechanisms can be preserved and administered without toxic effects, in either a liquid gel, an enteric coated capsule, or microencapsulated suspension, so that the antigens reach intestinal lymphoid tissues intact.

The present invention, employing a priming antigen as primer and a whole cell as an oral immunogen, also addresses toxicity and cross-reactivity issues, specifically directs induction of a mucosal response and eliminates the production of blocking antibodies.

Although experiments described in the parent application, Serial No. 07/965,916 showed r-Fbp to be an effective parenteral priming antigen prior to oral immunization with 340 WT cells, the subject experiments indicated that either polyphosphazene or a synthetic peptide were substantially better as priming agents prior to oral immunization with either live or irradiated PUT cells (Tables 1 and 5).

Use of a synthetic peptide as a parenteral priming antigen offers several advantages over the use of r-Fbp: 1) the relatively small peptide can be synthesized and purified by biochemical methods to yield a product free of endotoxin; 2) the synthetic peptide is more amenable to making amino acid substitutions that can be evaluated for improved priming capabilities; 3) the peptide is a more neutrally charged molecule than is the highly cationic r-Fbp (pl 10.35) and is thus less likely to affect DNA binding which is suspected to be a factor in certain types of autoimmune disease; and 4) the peptide primes better for PIII^" gonococci, which can be better as an oral vaccine candidate than the 340 WT cells.

The use of polyphosphazene as a priming agent is advantageous due to 1) its water-soluble, ionically-crosslinked structure and high molecular weight, which prevent excretion and allow for slow hydrolysis to effect its removal from tissue; 2) its potential as a universal primer for other oral vaccines; 3) its parenteral immunoactivity in relatively small doses (100 μg); and 4) its lack of parenteral toxicity. Killed cells and especially gamma-irradiated PUT cells offer several advantages as an oral immunogen: 1) anti-PIII or "blocking" antibodies are not induced due to the failure of this protein to be expressed by the deletion mutant; 2) gamma irradiation can enhance the oral immunogenicity of the PUT mutant (Table 1); 3) the irradiated PUT immunogen elicits a three- fold higher level of chamber protection and significantly better (P< 01) vaginal clearance of gonococci compared to live PUT cells or other immunogens (Table 1) ; and 4) gamma irradiation provides a convenient method of "in container" sterilization without toxic residues which are often left by other chemical methods of inactivation. This results in a safer product for oral immunization.

N. gonorrhoeae strain 340 WT was deposited on April 23, 1992 under the terms of the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852. Strain 340 WT has been assigned accession number ATCC 55320. The strain will be made available, without limitation, on the issuance of a patent.

The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES

Example 1. Comparison by chamber and vaginal clearance of various combinations of parenteral primers and oral components. To test different parenteral priming antigens as well as oral immunogens, more than 300 six-week old, female ICR outbred mice were injected intramuscularly with either 15 μg dose of recombinant iron-binding protein (r-Fbp) or with 20 μg/dose of a synthetic peptide (amino acid sequence DDQTYSIPSLFV, SEQ ID ΝO.T) seven times at weekly intervals. Parenteral priming was followed two weeks later by oral immunization with live or gamma-irradiated cells ofN. gonorrhoeae strain 340 wild type (340 WT) or a protein III deletion mutant of strain 340 (340 PIIT). Escherichia coli strain J5 cells were used as a control oral immunogen. Mice were orally immunized by giving ten weekly doses containing 10⁹ CFU in a volume of 0.5 ml by means of a gastric feeding tube.

Two weeks before the last oral immunization each mouse was surgically implanted with a subcutaneous culture chamber (Arko, R. J., J. Infect. Dis., 129:451 -455 (1974)).

Four weeks later, all groups of mice were given a graded dose challenge with virulent strain 340 WT cells. The infectious dose 50% (ID₅₀) was determined graphically for each group (Table 1).

In addition, a gonococcal vaginal clearance test was performed with each group. Mice were inoculated intravaginally with 400,000 CFU of virulent strain 340 WT cells and vaginal wash fluids were collected 6 h post-infection and assayed by culturing for gonococci (Table 1).

In previous experiments, r-Fbp-primed mice were protected to a greater degree following oral immunization with 340 WT cells, while mice primed with synthetic peptide were protected to a greater degree following oral immunization with 340 PUT cells. The data in Table 1 demonstrate that the combination of synthetic peptide primer and 340 PUT cell oral immunogen provided superior chamber protection as well as vaginal protection in comparison with the r-Fbp primer/ 340 WT cell oral immunogen combination. These data also show that gamma-irradiated 340 PIII" cells elicited a stronger immune response than live 340 PUT cells.

Example 2. IgA and IgG antibody titers in vaginal washings. To determine if immunization by the method of this invention yielded a mucosal immune response, IgA and IgG antibody titers were measured in vaginal wash fluids two weeks prior to vaginal challenge with gonococci. Mice were parenterally primed with seven weekly injections of either r-Fbp at 15 μg/dose or synthetic peptide at 20 μg/dose, followed by ten oral immunizations of 10⁹ CFU at weekly intervals. Titers were determined by whole cell ELISA on plates coated with 340 WT cells. The data in Table 2 indicate that the synthetic peptide primer and 340 PUT cells in combination as well as the r-Fbp primer and 340 WT cells in combination elicit production in the vagina of high titers of both IgA and IgG antibodies against 340 WT cells.

Example 3. Cross-reactive bactericidal activity of serum obtained from complement deficient mice.

Sera from mice parenterally primed with either purified iron binding protein (Fbp) or 340 WT cells alone, orally immunized with 340 WT cells alone, or parenterally primed with Fbp followed by oral immunization with 340 WT cells were assayed for bactericidal activity against a variety of microorganisms. A serum aliquot of 10 μl was placed over agar plates inoculated with confluent numbers of the following test organisms: N. gonorrhoeae 340 WT, N. gonorrhoeae 1 IB, N. gonorrhoeae F62 and N. meningitidis group A, and the percent of cells killed was calculated. As demonstrated in Table 3, the parenteral primer/oral immunogen combination method of immunization induced greater cross-reactive bactericidal activity than immunization with either component alone. Also, this response was induced in outbred ICR mice, which are complement deficient, indicating that the bactericidal activity was not totally dependent upon the classical complement pathway.

Example 4. Comparison of parenteral priming with peptide only and parenteral priming with peptide followed by oral immunization with whole cells.

Because the combination of synthetic peptide priming for oral immunization with 340 PUT cells provided superior chamber protection and vaginal protection as compared to the r-Fbp primer and 340 WT cell combination (Table 1), the synthetic peptide primer and 340 PIII^" cell combination has been examined further for its vaccine potential.

A group of 154 six-week old, female, ICR outbred mice was injected intramuscularly with 5, 20 or 50 μg of a synthetic peptide composed of the amino acid sequence DDQTYSIPSLFN (SEQ ID NO: 1) three, five or seven times at weekly intervals. In half of the mice, this parenteral priming was followed two weeks later by oral immunization with gamma-irradiated 340 PUT cells. Orally immunized mice were given ten weekly doses containing 10 CFU in a volume of 0.5 ml by means of a gastric feeding tube. Two weeks before the last oral immunization, each mouse was surgically implanted with a subcutaneous culture chamber, as described in Example 1.

Four weeks later, all mice were given a graded dose challenge with virulent strain 340 WT cells. The ID₅₀ was determined graphically for each group (Table 4). Mice primed with the synthetic peptide and orally immunized with gamma-irradiated 340 PHI^" cells resisted greater numbers of gonococci on challenge than did mice primed with synthetic peptide alone or oral immunogen alone.

In addition, the data in Table 4 indicate that priming with 50 μg of synthetic peptide for three or five weeks followed by ten oral immunizations with 340 PUT cells provides greater protection (highest ID₅₀) than that induced by priming with r-Fbp for seven weeks followed by ten oral immunizations with 340 WT cells (compare with Table 1).

Example 5. Comparison of soluble polyphosphazene and a gonococcal synthetic peptide as single injection primers for oral immunization.

A group of 63 six month old, female, ICR mice were injected intramuscularly with a combination of synthetic peptide (amino acid sequence DDQTYSIPSLFN, SEQ ID ΝO:l) and polyphosphazene or with either component alone in the doses listed in Table 5. This parenteral priming was followed four weeks later by oral immunization with gamma-irradiated 340 PIII^" cells. Oral immunization consisted of ten weekly doses of 10⁹ CFU in a volume of 0.5 ml administered by means of a gastric feeding tube. Two weeks before the last oral immunization, each mouse was surgically implanted with a subcutaneous culture chamber as described in Example 1.

Four weeks later, all mice were challenged with graded doses of virulent 340 WT cells. The ID₅₀ was determined graphically for each group and the percent of mice infected was calculated after challenge doses of 700, 5,000 and 92,000 CFU (Table 5).

The immune response in mice elicited by parenteral priming with either polyphosphazene (with or without peptide), a synthetic peptide or r-Fbp, followed by immunization with either 340 WT or 340 PIII^* cells was evaluated by determination of: 1) fD₅₀ in subcutaneous chamber challenge, 2) vaginal clearance, 3) IgG and IgA antibody production in the vagina, 4) cross-reactivity, and 5) bactericidal activity in the absence of complement.

These data indicate that strong immune responses (as measured by ID₅₀ levels in subcutaneous chambers and vaginal clearance of challenge gonococci) result from the parenteral administration of either polyphosphazene or a synthetic peptide primer, followed by oral immunization with 340 PHI^" cells (Tables 1, 4 and 5).

The combination of synthetic peptide and 340 PHI^" cells elicits the production of both IgA and IgG antibodies in the vagina (Table 2), which are important in providing the mucosal immunity required to prevent gonococcal colonization.

The application of polyphosphazene alone followed by oral immunization with

340 PIII^" cells protected against challenge infection significantly better than: 1) polyphosphazene and peptide combined, followed by 340 PHI^" oral immunization (P< 001), 2) peptide alone, followed by 340 PIII^" oral immunization (P<.05), and 3) 340 PIH^' oral immunization alone (P< 01).

The present invention also induced cross-reactive bactericidal activity (Table 3) against different gonococcal strains and protective immunity without the requirement for exogenous complement. Because the outbred ICR strain of mice used is deficient in the early complement component C2, the bactericidal antibodies and protection induced by parenteral immunization with whole gonococci in previous experiments required supplementation of the chamber fluid with an exogenous complement source in order to demonstrate this high level of immunity (Arko et al, J. Infect. Dis., 139:569-574 (1979)). These data show that the bactericidal activity demonstrated in these experiments was not totally dependent upon the classical complement pathway and that this invention can be more effective in providing protection at sites were complement components are limited (e.g., the urogenital tract). Example 6. Oral immunization of mice for cross protective immunity to N. gonorrhoeae and N. menineitidis.

Animal model, immunization, and challenge. Outbred Institute of Cancer Research (ICR) female mice of various age groups (2-12 months) obtained from Harlin Sprague-Dawley, Indianapolis, IN were orally immunized at weekly intervals with 0.5 ml doses (10⁹ CFU) of live or τ-irradiated N. gonorrhoeae or N. meningitidis cells by using a syringe and a 18 ga intragastric feeding needle. After 5-10 oral doses of vaccine, each mouse was surgically implanted with a porus stainless steel chamber by methods previously described (1). At approximately 4-5 weeks following implantation, each chamber along with chambers in nonimmunized controls were challenged by administration, via a 25 ga needle and syringe, of various amounts of CFUs of live N. gonorrhoeae or N. meningitidis according to the graded-dose challenge method described below. At 48 to 72 h intervals after challenge, 1-5 μl chamber fluid specimens were aspirated from each mouse by syringe and 25 ga needle and streaked directly onto agar plates of GC base with IsoVitaleX (BBL, Cockeysville, MD). Culture plates were incubated at 36°C under 5% CO₂ for 24-48 h and then examined for bacterial growth. Representative colonies were tested for reactions with oxidase, catalase, and gram stain reagents as required for identification. Mice with culture negative chambers were reinoculated with 10-fold increases in challenge CFU until infection resulted or greater than 10⁵ CFU were resisted. Mice that became culture positive were recultured at 3-7 day intervals to determine the duration of infection. The challenge dose ΪD_S0 (determined graphically) and the median clearance rate of infection (CR₅₀) were used to determine the relative resistance induced by various oral immunizations. Statistical comparisons were made by using the Chi-square method.

Parenteral priming agents. The purified 37 Kd neisserial iron binding protein (Fbp) was obtained as a gift from Dr. Tim Mietzner, University of Pittsburgh, PA. (7) Mice were injected im with 15 μg of Fbp for 7-10 times at weekly intervals and starting two weeks after the final injection given a series often weekly oral immunizations with N. gonorrhoeae cells. The N. gonorrhoeae protein IB outer membrane peptide DDQTYSIPSLFV described by Heckels et al. (8) was synthesized by FastMoc™ chemistry on an ABI Model 430 peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) according to manufacturer's protocols. Mice were injected with 20 μg of synthetic PIB peptide (SP) for seven times at weekly intervals or given a single injection of SP in various amounts (50-500 μg) at two weeks before starting oral immunizations.

A water soluble ionically-crosslinked polyphosphazene (PPP) was obtained from Virus Research Institute, Cambridge, Massachusetts and used as a single dose nonbacterial primer. Mice were injected i.m. with 100 μg of PPP two weeks before starting oral immunization with N. gonorrhoeae cells.

A commercial Haemophi/us inβuenzae b conjugate vaccine (Hib-dt, diphtheria toxoid conjugate, ProHIBit®, Connaught Laboratories, Inc., Swiftwater, PA) was tested as a single dose primer. Mice were injected intramuscularly (im) with 0.1 the human dose of vaccine one week before starting a series of oral immunizations.

Oral adjuvants. The E. coli labile toxin (LT) (Berna Products Corporation, Coral Gables, FL) was tested as an oral adjuvant by mixing 10 μg of LT with each 0.5 ml dose ofN. gonorrhoeae or N. meningitidis cells.

Lithium chloride (Sigma Chemical Co., St. Louis, MO) and taurine (2-amino ethanesulfonic acid, Sigma Chemical Co., St. Louis MO) were each tested as oral adjuvants by adding 5 mg to each 0.5 ml dose of τ-irradiated cells.

C3d conjugated cells. The C3d complement peptide (KNRWEDPGKQLYNVEA, SEQ ID NO:5) (Sigma Chemical Co., St. Louis, MO) was coupled with glutaraldehyde (0.5% v/v) to C4 deficient gamma irradiated meningococcal cells at a concentration of 0.5 mg/ml of cells (1.5xl0⁹CFU). Cells were washed in PBS and resuspended to lxl 0⁹ CFU/ml and used to orally immunize mice (0.5 ml/dose) given weekly for ten weeks.

Bacteria and Immunogens. Cells used for preparing oral immunogens were grown on plates of GC base agar supplemented with 1% Isovitalex for 18-20 h under 5% CO₂ at 36°C. Cells were harvested into PBS (pH 7.2) by using a cotton-tipped applicator stick and were photometrically adjusted to a turbidity containing approximately 10⁹ CFU/ml. Cell suspensions were inactivated by exposure to Cobalt 60 (1650-2500 Grays) in a Model 232 Gamma Cell. Each mouse was orally immunized weekly with 0.5 ml of a τ-irradiated cell suspension for up to ten weeks.

Additional N. gonorrhoeae and N. meningitidis strains used for testing cross- protection in immunized mice were obtained from culture collections maintained at CDC Laboratories. Various protein, polysaccharide, and LOS typing was determined according to published procedures (Wedege et al., 1990. J. Med. Microbiol 31:195- 210). N. meningitidis strain FAM- 18 tested: C:2a,5 :P 1.2:L3,7,9. The N. meningitidis group B isolate tested: B:2b:P2.3 and the N. meningitidis group A isolate tested: A:21 :P1.10:L10. The N. gonorrhoeae 340 isolate was nonreactive with the N. meningitidis typing sera but tested as an D3-6 in the gonococcal typing procedures (Knapp et al., 1984. J. Infect. Dis. 150:44-48).

Serology. Blood specimens (400 μl) obtained by cardiac puncture from mice under CO₂ anesthesia were allowed to clot and serum was removed after centrifugation. Whenever possible, specimens were tested from individual mice or as pools consisting of equal volumes of serum from three mice. Specimens were obtained one week before challenge of control and immunized mice and were stored at -70 °C until tested.

' Whole cell ELISA using N. gonorrhoeae or N. meningitidis cells. Piliated cells of gonococcal strain 340 (wild type) were grown overnight (16-20 h) on described GC plates in a CO₂ incubator. Cells were suspended in PBS pH 7.2 and the optical density adjusted to Abs^ = 0.1 (ca.3xl0⁶ CFU/ml). One hundred microliters of the whole cell suspension was added into each well of a microtiter plate (Immulon II) and centrifuged at 2000 rpm for 15 min before adding 50 μl of 0.5% glutaraldehyde. The plate was held at room temperature for 1 h. Wells were washed three times with PBS, 100 μl of blocking solution (PBS + 3%BSA + 0.1% Tween-20) was added to the wells and the plate was incubated at room temp for an additional hour. Blocking solution was also used in making appropriate dilutions of test samples (serum, chamber fluid, or vaginal wash) and incubation was carried out overnight at room temperature. The wells were washed and a dilution of 1 :1000 anti-mouse IgG/M/A conjugated with horseradish peroxidase (100 μl/well) was added. The plate was incubated at 37°C for one hour before developing with substrate TMB. The color reaction was stopped w/ IN H₂SO₄ and read at OD_4J0.

Serum Bactericidal tests. A previously described microtiter assay using baby rabbit serum as complement and the meningococcal group C, M-l 1 test organism was employed to determine bactericidal antibody levels in mouse sera (13).

Experiments were conducted with PIII- N. gonorrhoeae strain 340 whole cells, C4- N. meningitidis FAM-18 whole cells or wild type whole cells as immunogens. As shown in tables (2-8) substantial levels of strain specific protection, as well as cross- protection, were observed in N. meningitidis-C orally immunized mice. Cells ofN. gonorrhoeae strain 340 in which PIII had been deleted were substantially more effective as an oral immunogen that were wild type N. gonorrhoeae strain 340 cells. Like the N. gonorrhoeae 340 PIII^" cells, oral immunization with the C4^" N. meningitidis cells was also shown to be significantly (P<.01) more protective than was observed with isogeneic wild type cells (Table 6). Also, high levels of protection were observed in polyphosphazene-primed mice given N. meningitidis orally.

The primary cross-challenge ofN. meningitidis-C orally immunized mice with N. meningitidis- A produced a relatively low (≤30 CFU) chamber DD_J0. However, the clearance rate for N. meningitidis- A in the immunized group (six days) was about twice that observed in nonimmunized controls (13 days). In addition, 14% (2/14) of the immunized mice resisted the highest N. meningitidis- A challenge dose given (3,000 CFU) while all six^' control mice were infected by a 100-fold lower challenge. The variation in response of some immunized mice probably reflects their outbred genetic background.

As shown in Table 7, a high level of cross-protection against N. meningitidis- A (ID₅₀ >2.2 x 10⁵ CFU) and N. meningitidis-B (ID_J0> 5.7 x 10⁶ CFU) was obtained in N. meningitidis-C orally immunized mice that had previously resisted N. meningitidis-C challenge and were later rechallenged with N. meningitidis- A and N._. meningitidis-B. This suggests that subseαuent environmental exposure to neisseria might plav a role in boosting the immune response following oral immunization with related neisserial strains. Table 8 summarizes the protective effects demonstrated in the experiments described in Table 7.

Mice primed by im injection of polyphosphazene and given E. coli LT adjuvant mixed with the oral bacterin showed similar resistance to challenge with N. meningitidis- C as did mice that received oral N. meningitidis-C cells alone (Table 7). However, upon rechallenge of these mice with a N. meningitidis- A strain, significantly (P< 05) less protection was observed in mice given polyphosphazene priming two weeks before initiating oral immunization (Table 7).

As shown in Tables 9-11, various oral adjuvants as well as a parenteral priming injection with Hib-dt vaccine were evaluated for enhancement of oral immunization. When these groups were challenged with N. meningitidis FAM-18 wild type cells, mice parenterally primed with the Hib-dt vaccine were significantly (P=0.02) more resistant compared to nonimmunized controls. This group also had one of the highest median serum bactericidal antibody (SBA) responses measured in serum drawn one wk prior to challenge (Table 11). The median ratio of serum group C anticapsular IgAl to IgG was also highest in this group. Two less protected groups receiving taurine as an oral adjuvant or the combined N. meningitidis serogroups B+C showed significantly (>fourfold) less SBA as well as a lower median IgAl to IgG ratio. A group that received the E. Coli LT oral adjuvant along with N. meningitidis C4^" cells also developed a high median SBA (1 :2048) even though 50% (3/6) mice became infected upon primary challenge. However, the median level of chamber colonization at 48 hours after challenge was much lower, with only one CFU, as compared to >300 CFU/5 μl in nonimmunized controls.

The group that received oral lithium along with C4^"N. meningitidis cells demonstrated the best overall resistance to challenge when results from both N. meningitidis and N. gonorrhoeae challenges were evaluated (Table 11).

The challenge results shown in Tables 9-11 were obtained from five groups of mice given identical oral N. menin^gitidis C4^" immunization but with different oral or 32 parenteral adjuvants. Each group was then divided and challenged with virulent N. meningitidis or N. gonorrhoeae wild type cells. When results from both primary challenges were combined, the infection rate for mice orally immunized with N. meningitidis C4^" cells plus lithium chloride was 4/16 (25%), as compared with 17/21 (81%) for nonimmunized controls, which is a highly significant (P<0.001) difference. The only other group in which the combined rate of 3/11 (27.3%) was highly significant had received a parenteral im injection of Hib-dt vaccine 1 week before starting the oral regimen. The combined infection rate for mice that were orally immunized with N. meningitidis C4^" cell without adjuvant was 7/16 (43.8%). The combined results from the two oral adjuvants, taurine and E. coli LT, was an infection rate of 16/30 (53.3%). The combined results from the two oral adjuvants, parenteral Hib-dt and oral lithium chloride, was 7/27 (26%) (Tables 9-11).

In experiments with 12-mo-old mice (Tables 9-11) beneficial effects appeared to result from the addition of either Hib-dt or lithium chloride as oral adjuvants. Priming older mice by a single im injection of a Hib-dt vaccine one week before the start of oral immunization also gave significantly (P=0.02) better protection to N. meningitidis challenge (with 16.6% of mice infected), compared to 77.7% infection of nonimmunized controls (Table 9). Although the effects of priming mice with an antigenically unrelated parenteral vaccine prior to giving oral immunization has not been determined it is likely that these primers induce acute phase serum proteins i.e. lipopolysaccharide binding protein (LBP) or LPS receptors (CD 14) as well as cause the proliferation of cytokine producing T-cells that may play a role in enhancing and/or "switching" the immune response to subsequently given oral vaccines.

The highest level of protection against N. meningitidis challenge in older mice was found in a group that was first primed with a parenteral Hib-dt vaccine before starting oral immunization. When sera collected 1 week before challenge were analyzed for serum bactericidal activity against N. meningitidis-C strain M-l 1, a median titer of 1 :2048 was found. Further analysis of sera from these orally immunized groups revealed that the Hib-dt primed mice had the highest median ratio of group C anticapsular IgAl to IgG (1 : 128) compared to mice in other groups that were less protected. One group that received taurine as oral adjuvant along with oral immunization showed a significantly lower median serum bactericidal titer (1 :64) as well as a lower median IgAl to IgG ratio (1: 16) and were less protected against N. meningitidis or N. gonorrhoeae challenge. The other group to show a similar pattern of serum antibody activity received an oral combination ofN. meningitidis- and N. meningitidis-C τ-irradiated cells mixed 1 : 1. This group also showed a 50 percent infection rate after a modest (43 CFU) challenge with group C meningococci (Tables 9 and 11).

The data set forth in Tables 13-15 further demonstrate the protective affect against N. meningitidis Group B provided by immunization with the vaccine of the present invention.

These results show that oral immunization of mice with τ-irradiated N. meningitidis-C, C4-deficient cells provides substantial protection against challenge with homologous N. meningitidis group C, as well as N. meningitidis group A, N. meningitidis Group B and the strains ofN. gonorrhoeae 340. Because of the need for meningococcal immunization in early childhood, the whole cell immunogen of the present invention could be given orally for this purpose while simultaneously increasing resistance to N. gonorrhoeae infections that might be encountered later in life.

Example 7. Human vaccination.

A regimen for administration of either the gonococcal or meningococcal vaccine to humans can include a single parenteral injection of up to 800 μg of priming antigen (e.g. polyphosphazene, tucaresol) followed four weeks later by oral administration of enteric coated capsules at one week intervals for ten weeks. Each capsule can contain 5xl0⁹ CFU of gamma-irradiated 340 PIII^' gonococci or C4- meningococci.

A regimen for the oral administration of C4- N. meningitidis whole cells can include oral administration of between lxlO⁹ and 8xl0⁹ CFU/ml and most preferably 5xl0⁹ CFU/ml in a pharmaceutically acceptable carrier at one week intervals for up to ten weeks and most preferably four to six weeks. The whole cells can be contained in enteric coated capsules containing 5x10⁹ CFU of gamma-irradiated C4- N. meningitidis or a combination of C4- N. meningitidis and PIII- N. gonorrhoeae with or without an oral adjuvant such as lithium chloride (5 mg with each dose of τ-irradiated cells) or tucaresol (10-20 mg/kg simultaneously with each dose of cells or up to two days prior to initial administration of cells).

Human subjects for controlled gonococcal experiments can be selected according to established protocols (see, e.g., Cohen et al., 1994. J. Infectious Diseases 169:532-537). Preimmunization medical histories including prior gonococcal or meningococcal illness as well as pharyngeal and urethral bacterial cultures can be obtained. Pre and post immunization serum samples can be obtained at appropriate intervals and tested for complement-mediated bactericidal activities against gonococcal and/or meningococcal test strains. At six to ten weeks following the final oral dose of vaccine, controlled challenges with gonococci can be performed. The challenge dose ID_JO can be determined for each individual and compared with results obtained in placebo immunized matched controls.

Efficacy results of the present invention regarding protection against meningococcal and gonococcal diseases can also be obtained through clinical field trials. A geographical area where a high rate of meningococcal disease is present can be selected. The oral vaccine can be administered to a statistically valid number of subjects in various age groups and appropriate pre and post immunization specimens can be obtained, as described above. In addition, the rate of meningococcal carriage can be determined by taking appropriate nasopharyngeal swabs, as would be known to one of ordinary skill in this art. The test subjects can be monitored over an extended period of time for subsequent meningococcal and/or gonococcal diseases. Comparison of disease rates and carriage rates between orally immunized and matched (non-immunized) controls can be used to determine the efficacy of the present vaccine, according to protocols well known in this art.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

Table 1. Comparison of protection, as measured by subcutaneous chamber and vaginal clearance models, in parenterally primed and orally immunized mice.

1. Synthetic peptide DDQTYSIPSLFV (SEQ ED NO:l) injected at 20 μg per mouse for seven weekly doses. Same for recombinant iron binding protein (r-Fbp) at 15 μg/dose.

2. Live or gamma-irradiated (2.5 x 10⁵ rads) 340 WT or 340 PUT cells administered orally at 10⁹ CFU per mouse for ten weekly doses.

3. Intravaginal inoculation of 400,000 CFU of live 340 WT. Vaginal wash fluids were collected 6 h post-infection. Significance determined by Chi square analysis by comparison with clearance in virgin control mice.

4. One mouse expelled subcutaneous chamber prior to challenge.

37

Table 2. Median antibody titers to N. gonorrhoeae¹ in vaginal wash from normal and immunized mice obtained two weeks before vaginal challenge with gonococci².

1. Median titer of nine mice collected in three pools of vaginal wash and tested by whole cell ELISA on plates coated with 340 WT.

2. Mice immunized by seven parenteral priming injections followed by ten oral immunizations of 10 colony forming units at weekly intervals.

3. Virgin control mice.

38

Table 3. Bactericidal activity of serum obtained from mice primed and/or orally immunized with different immunogens ofN. gonorrhoeae .

Percent killing produced by application of 10 μl aliquots of serum over agar plates inoculated with confluent numbers of microorganisms.

A) Mice orally immunized with whole cells of strain 340 WT.

B) Mice parenterally primed with iron-binding protein (Fbp) followed by oral immunization with whole cells of 340 WT.

C) Mice parenterally immunized with Fbp.

D) Mice parenterally immunized with whole cells of 340 WT.

39

Table 4. Comparison of ID₅₀ in subcutaneous chambers of mice given different regimens of parenteral priming alone or in combination with oral immunization^1,2.

1. ID₅₀ determined graphically from data obtained following graded dose challenges of 5- 10 mice per group.

2. Mice were orally immunized weekly with 10⁹ CFU of 340 PIII^" for ten weeks.

3. Synthetic peptide DDQTYSIPSLFV (SEQ ID NO: 1) injected intramuscularly in 0.1 ml volume of phosphate buffered saline, pH 7.4.

4. ^"ID₅o of virgin control mice was 180 CFU.

Table 5. Animal Challenge Experiments Comparing Soluble Polyphosphazene and a Gonococcal Synthetic Peptide¹ as Single Injection Primers for Oral Immunization.

o

1. Amino acid sequence DDQTYSIPSLFV (SEQ ID NO: 1).

2. Results of culture of specimens taken from subcutaneous chambers in mice challenged with 340 WT cells 48 hours earlier.

Table 6. Primary and Secondary Challenge results in mice orally immunized with either τ-irradiated cells of the wild type or protein class 4 deficient N. meningitidis FAM-18.

Orally immunized mice were first challenged with N. meningitidis and mice shown to be culture negative after the 2000 CFU challenge were rechallenged with graded doses ofN. gonorrheae.

Number of mice culture positive in chamber fluids over number challenged.

Table 7. Challenge results for meningococcal Group C orally immunized mice¹

Group C Challenge CFU³ Rechallenge with Group A Rechallenge with Group B

l im lO wks

Rx Prime⁵ Oral 15 290 2800 94,000 1 x 10' 380 29,000 2.2 x 10⁵ 1.77 x 10³ 5.7 x 10*

I None Mc-C4- 0/8 1/8 1/8 2/8 2/8 1/8 1/8 2/8 1/8 1/8 π None Mc-C4- 0/8 0/8 1/8 1/8 1/8 2/8 2/8 2/8 2/8 3/8 +LT lO g

III PPP Mc-C4- 0/17 0/17 4/17 7/17 9/17 5/10 5/10 5/10 7/17 9/17 lOOμg rv PPP Mc-C4- 0/10 0/10 0/10 2/10 3/10 5/9 5/9 5/9 3/10 6/10

V⁴ None None 3/9 6/9 8/9 9/9 NT 3/9 8/9 8/9 2/11 10/11

Group C FAM-18 made C4- by insertional inactivation of C4 gene and used as γ-irradiated cells (10' CFU) for 10 weekly oral immunizations.

Single intramuscular (IM) injection of polyphosphazene three weeks before start of oral immunization.

Challenge initiated one week following last oral immunization or one week after previous challenge. Only culture negative mice were rechallenged with meningococcal groups A and B. Results expressed as number of mice culture positive/number tested at 48 hours following chamber challenge.

Challenge controls for meningococcal groups A, B and C were not previously infected by other serogroups.

Table 8. Summary of protective effects of oral immunization with Group C, Protein 4 deficient (C4-), γ-irradiated cells of Neisseria meningitidis determined by chamber challenge of mice¹ with meningococcal Groups A, B, and C.

1 x im 10 wks No. Protective Protective

R_x Prime Oral Mice ID*- McC Index C ED^ McA Index A ID_™ McB Protective Index B

I None Mc-C, >10⁶ CFU >6000 >2.2 x 10' >1375 5.7 x 10' >5.1 C4- π None Mc-C, >10⁴ CFU >6000 >2.2 x 10⁵ .1375 =>5.7 x 10' >5.1

C4- + LT lO g in PPP Mc-C, 17 6xl0⁵ CFU 3750 380 0.2 1.8 x 10" 1.6 lOOμg³ C4- rv PPP Mc-C, 10 >10⁶ CFU >6000 <380 <0.2 1.7 x 10' 1.5 lOO g C4- + LT lO g

None None 160 CFU 1.0 1600 1.0 1.1 x 10* 1.0

Group C FAM-18 N. meningitidis made C4- by insertional inactivation of P4 gene.

Female ICR outbred mice were used for immunization and challenged in subcutaneous chambers with graded doses of Group A, B, or C meningococci

Water soluble polymer of polyphosphazene.

Table 9. Neisseria meningitidis Challenge results from parenterally primed and/or orally immunized twelve month-old female mice

Primary Challenge* Infection Rate Median level'¹

Immunization; and Percentage of Colonization

Primer Hib-dt' Lm. 1 wk before oral Nm-C l OX weekly 1/6 (16.6) 100 CFU

Primer PBS control' Lm. 1 wk before Oral

Nm-C, 10X weekly 5/9 (55.5) >300 CFU

Oral Nm-C + 5 mg of Lithium Cl, 10X in (28.5) >300 CFU

Oral Nm-C + 5 mg

Taurine, 10X 4/8 (50.0) >300 CFU

Oral Nm-C + 10 μg of E. coli LT adjuvant, 10X 3/6 (50.0) 1 CFU

Non-immunized controls 7/9 (77.7) >300 CFU

N. meningitidis FAM-18 wild type challenge in subcutaneous chambers with 43 CFU of live cells two weeks after the final oral.

Hib-dt vaccine (Connaught, Swiftwater, PA) given as a parenteral primer (0.1 human dose) one week before starting oral immunization with 10' CFU of τ-irradiated N. meningitidis C4" deficient celk).

Sterile phosphate buffered saline pH 7.2 as primer control.

Median number of CFU grown from 5 μl of chamber fluid taken 48 h after challenge. Table 10. Neisseria gonorrhoeae challenge results from parenterally primed and/or orally immunized twelve month-old female mice

Primary Challenge*

Immunization: Infection Rate Median level and Percentage of Colonization*

Primer Hib-dt^b Lm. 1 wk before oral Nm-C, 10X weekly 2/5 (40) >300 CFU

Primer PBS control' Lm. 1 wk before Oral

Nm-C, 1 OX weekly in (29) 200 CFU

Oral Nm-C + 5 mg of Lithium Cl, 10X 2/9 (22) >300 CFU

Oral Nm-C + 5 mg

Taurine, 10X 4/7 (57) >300 CFU

Oral Nm-C + 10 μg of E. coli LT adjuvant, 10X 5/9 (55) >300 CFU

Nonimmunized controls 10/12 (83) >300 CFU

N. gonorrhoeae strain 340 wild type challenge in subcutaneous chambers with 600 CFU of live cells two weeks after the final oral.

Hib-dt vaccine (Connaught, Swiftwatcr, PA) given as a parenteral primer (0.1 human dose) one week before starting oral immunization with 10' CFU of τ-irradiated N. meningitidis CA' deficient cells.

Sterile phosphate buffered saline (PBS) pH 7.2 as primer control.

Median number of CFU grown from 5 ul of chamber fluid taken 48 h after challenge.

Table 11. Results of Neisseria meningitidis group C serum bactericidal and ELISA IgAl and IgG anticapsular antibody tested in orally immunized mice*

Combined Results

Immunization: Median serum Mc Median IgAl for Gc and Mc bactericidal titer to IgG ratio challenges*

Primer Hib-dt

Lm. 1 wk before oral

Nm-C for lOx weekly 1:2048 1:128 3/11 (27.3%)

PBS primer control

+ oral Nm-C lOx weekly 1: 1024 1:64 7/16 (43.8V.)

Oral Nm-C + 5 mg lithium Cl lOx weekly 1:2048 1:64 4/16 (25%)

Oral Nm-C + 5 mg

Taurine 1 Ox weekly 1:64 1:16 8/15 (53.3%)

Oral Nm-C + 10 μg

E. coli Lt adjuvant lOx weekly 1:2048 1:64 8/15 (53.3%)

Oral Nm-B+

Nm-C(l:l) 1:380 1:32 5/10 (50%)

Non immunized controls <1:8 NT 17/21 (81.0%)

Serum collected 1 week before challenge.

Challenged with 43 CFU of Nm-C or 600 CFU of Gc 340 wild type cells and infection determined by culture of subcutaneous chamber 48 h later. Number infected over number challenged.

Table 12. Effects of gamma irradiation used for cell inactivation on the immunogenecity of a protein class 4 deficient meningococcal whole cell vaccine tested for protection against gonococcal challenge in a mouse subcutaneous chamber model.

Irradiated with cobalt-60 for 5, 15, 45 or 85 minutes, dosages expressed in Grays

Fourteen month-old, female, ICR white mice challenged with Neisseria gonorrhoeae strain 340 two days after receiving the tenth oral immunization with 10⁹ colony forming units of irradiated meningococcal cells.

Infectious dose 50 percent determined graphically.

Table 13. Resistance of orally immunized* and previously challenged¹* mice to rechallenge with Group B meningococci

Weekly oral immunization with 10⁹ CFU of gamma irradiated protein C-4 deficient Group C meningococci (FAM-18)

First challenge with Gc strain 340 or Mc-C strain FAM-18 4 wks followingg the final dose of oral vaccine

Rechallenge of subcutaneous chambers with 4000 CFU of wild type Group B meningococci 3 wks following clearance of the first challenge

Table 14. Comparison of meningococcal group B challenge results in subcutaneous chambers' of mice given previous (A) group C oral immunization (B) previously infected challenge controls'¹ or (C) virgin challenge controls

Subcutaneous chambers challenged with 4000 CFU of Neisseria meningitidis Group B 2 wks following clearance of their previous challenge or 7 wks following their last oral immunization with 2-10 weekly innoculations of 10⁹ CFU of gamma irradiated Group C, protein class 4 deficient meningococci.

Previous self-terminating infection with Neisseria gonorrhoeae strain 340 or with a Group C meningococcal strain FAM-18.

Table 15. Results from Neisseria meningitidis rechallenge of mice orally immunized with gamma-irradiated, group C meningococcal cells and (A) resisted primary challenge with either N gonorrhoeae strain 340 or with N. meningitidis group C strain FAM-18 or (B) became transiently infected when challenged with either organism which after being cleared for at least 1 wk where rechallenged in subcutaneous chambers with a serogroup B meningococcal strain.

Oral immunization with 10⁹ CFU of protein class 4 deficient meningococcal cells.

Challenged with up to 1.5 x 10* CFU of wild type gonococci or 5 x 10^s CFU of group C meningococci.

Persistence of infections in nonimmunized controls was 1-2X longer in duration than observed in orally immunized mice.

Rechallenge of subcutaneous chambers with 4 x 10³ CFU of serogroup B meningococci (12/12 virgin controls infected).

Number infected/number at risk determined by culture analysis of chamber fluids at 48h post challenge. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Arko, Robert J. Chen, Cheng-Yen Morse, Stephen A. Trees, David

(ii) TITLE OF INVENTION: IMMUNIZATION AGAINST NEISSERIA GONORRHOEAE AND NEISSERIA MENINGITIDIS

(iii) NUMBER OF SEQUENCES: 5

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: NEEDLE & ROSENBERG, P.C.

(B) STREET: 127 Peachtree Street, NE, Suite 1200

(C) CITY: Atlanta

(D) STATE: Georgia

(E) COUNTRY: USA

(F) ZIP: 30303-1811

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/475,531

(B) FILING DATE: 07-JUN-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Perryman, David G.

(B) REGISTRATION NUMBER: 33,438

(C) REFERENCE/DOCKET NUMBER: 14114.0207

(ix) "TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (404) 688-0770

(B) TELEFAX: (404) 688-9880

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

Asp Asp Gin Thr Tyr Ser lie Pro Ser Leu Phe Val 1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gin His Gin Val Tyr Ser lie Pro Ser Leu Phe Val 1 5 10

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Glu His Gin Val Tyr Ser lie Pro Ser Leu Phe Val 1 5 10

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Ala Ser Val Ala Gly Thr Asn Thr Gly Trp Gly Asn Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Lys Asn Arg Trp Glu Asp Pro Gly Lys Gin Leu Tyr Asn Val Glu Ala 1 5 10 15

Claims

What is claimed is:

1. A method for protecting a human patient against infection by Neisseria meningitidis and Neisseria gonorrhoeae comprising orally administering to the patient a protective amount of a class 4 protein-deficient, killed whole cell of Neisseria meningitidis.

2. The method of claim 1, wherein the class 4 protein-deficient killed whole cell of Neisseria meningitidis is produced from Neisseria meningitidis strain FAM-18.

3. The method of claim 1, wherein the whole cell of Neisseria meningitidis is killed by gamma irradiation.

4. A method for protecting a human patient against infection by Neisseria meningitidis and Neisseria gonorrhoeae comprising orally administering to the patient a protective amount of a class 4 protein-deficient, killed whole cell of Neisseria meningitidis and an oral adjuvant.

5. The method of claim 4, wherein the oral adjuvant is lithium chloride.

6. The method of claim 4, wherein the class 4 protein-deficient killed whole cell of Neisseria meningitidis is produced from Neisseria meningitidis strain FAM-18.

7. The method of claim 4, wherein the whole cell of Neisseria meningitidis is killed by gamma irradiation.

8. A kit comprising a container with a class 4 protein-deficient, killed whole cell of N. meningitidis, in a pharmaceutically acceptable carrier for oral administration.

9. A kit comprising a first container with a class 4 protein-deficient, killed whole cell ofN. meningitidis in a pharmaceutically acceptable carrier for oral administration and a second container with an oral adjuvant in a pharmaceutically acceptable carrier for oral administration.

10. The kit of claim 9, wherein the oral adjuvant is lithium chloride.

11. A method for protecting a human patient against infection by Neisseria meningitidis and Neisseria gonorrhoeae comprising parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response and subsequently orally administering a protective amount of a class 4 protein-deficient, killed whole cell of Neisseria meningitidis.

12. A method for protecting a human patient against infection by Neisseria meningitidis and Neisseria gonorrhoeae comprising parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response and subsequently orally administering a protective amount of a Protein Ill-deficient killed whole cell of Neisseria gonorrhoeae.